Keywords

Photoreceptor; Dual-luciferase reporter; PRPF31
mutation; TFPT

Introduction

PRPF31 encodes the ubiquitous splicing factor PRPF31, an essential
component of the U4/U6.U5 tri-snRNP. The gene is highly conserved
throughout evolution, with orthologues in all vertebrate species,
invertebrates and lower species, including yeast. Mutations in PRPF31 have been shown to be a major cause of autosomal dominant retinitis
pigmentosa (adRP), accounting for 5% of disease in the UK [1,2].

A unique feature of PRPF31-associated adRP is phenotypic nonpenetrance,
where within affected families there are asymptomatic
mutation carriers. This is due to the existence of differentially expressed
wildtype PRPF31 alleles, with co-inheritance of a PRPF31 mutation
and a higher-expressing allele providing protection against clinical
manifestation of disease. It has been shown that there is variable
expression of PRPF31 in the general population and, that within
mutation carrying families, asymptomatic mutation carriers have more
than two-fold higher expression levels of wildtype PRPF31 compared
to symptomatic individuals [3-6].

One study looked at phenotypic discordance between mutationcarrying
siblings and observed that the symptomatic and asymptomatic
siblings consistently inherited different wildtype chromosome 19q13
alleles from the non-mutation carrying parent [7]. It is generally
thought, therefore, that cis-acting factors that affect the level of PRPF31 expression (such as regulatory region polymorphisms) underlie
phenotypic non-penetrance in mutation-carrying families. However,
attempts to identify such changes have not yet been successful.

It has also been demonstrated that there is increased expression of both PRPF31 alleles in asymptomatic mutation-carrying individuals,
with subsequent degradation of the mutant molecule by nonsense
mediated decay – this indicating that at least one factor that alters PRPF31 expression acts in trans [8]. One possible trans-acting factor
was identified through the association of higher PRPF31 expression and
an expression quantitative trait locus (eQTL) at 14q21-23, although the
exact factor was not characterized [3]. It was also shown that variable
expression of CNOT3 is an important factor in determining PRFP31 expression level - with increased levels of CNOT3 protein causing
transcriptional repression of PRPF31 [9]. CNOT3 is a component of
the Ccr4-Not transcription complex, which is a global regulator of
RNA polymerase II-mediated transcription [10].

Attempts to generate mouse models of PRPF31-associated adRP
have failed to yield animals with a retinal degeneration phenotype.
Neither Prpf31 knock-in animals nor knock-out animals displayed
retinal degeneration, and the animals did not have any visual defect
at up to 18 months of age [11]. There is some evidence that Prpf31 knockout mice develop changes within the retinal pigment epithelium
(RPE), with vacuolation, loss of the basal infoldings and accumulation of amorphous deposits between the RPE and Bruch's membrane [12].
There was not, however, death of retinal photoreceptor cells (the
primary histological and pathological change in human disease) and no
change in retinal function was reported – and so these animals cannot
be considered a model for human disease [12]. It is possible that the
same RPE changes are observed in asymptomatic individuals, but this
study is not feasible. It can be concluded that in mouse, 50% of protein
level is sufficient for normal retinal function.

Regulation of gene expression is central to pathogenesis of PRPF31 mutations in humans and also the failure of animal models of disease
and it is necessary, therefore, to understand the 5’ architecture of
the PRPF31 gene. PRPF31 and TFPT are arranged in a bidirectional
gene pair, with partially shared exon 1, at chromosome 19q13.4. It is
increasingly recognised that many genes exist in bidirectional pairs,
which are defined as two genes that lie in a head-to-head arrangement,
on opposite DNA strands, with less than 1kb separating their
transcription start site (TSS).

TFPT, also known as CF3 fusion partner or FB1, encodes a 253
amino acid protein, that was first identified in some cases of paediatric
pre-B-cell acute lymphoblastic leukaemia as the fusion partner of the
transcription factor E2A [13]. A role for the human protein has not yet
been described, but the rat homologue, Tfpt, has been shown to be proapoptotic
and might modulate cerebral apoptosis [14].

As differential expression of PRPF31 underlies phenotypic nonpenetrance,
a study was undertaken to characterize the core promoter
element of the gene (and the bidirectional gene pair, TFPT), as it was
considered important to understand the transcriptional regulation of
the gene in the normal population [15]. The work by Rose et al. [15]
repeated and extended a previous promoter characterization study by
Brambillasca et al. [16], which had assayed fragments from the reverse
strand in order to characterize a putative TFPT promoter element. Dual
luciferase reporter assay was performed and a fragment termed BiP was
defined as the core promoter of PRPF31, whereas the core promoter of TFPT was defined as a fragment termed P.31-Luc [15].

It was considered that studying the conservation and evolution
of the PRPF31 and TFPT core promoters in several mammalian
species might shed light on the complex regulation of these genes
and the failure of mouse models of PRPF31-adRP. The present study
design was based on the results of the study on the human genes [15],
through the identification of regions homologous to the active human
DNA fragments. The homologous genomic regions were tested by
dual-luciferase reporter assay in order to assess conservation of gene
regulation. Where homology with the human region was found to be
low, novel fragments were assayed.

Methods

Bioinformatic analysis

Evolutionary conservation of regions was analyzed using ECR
browser in NCBI DCODE software suite using default software settings
for each program [17].

PAZAR transcription factor work space was used to find TFBS for PRPF31 and TFPT promoters in human and monkey and bidirectional
promoters of dog and mouse [18]. Transfac, Jaspar and Oreganno
vertebrate profiles were used to define TFBS in conserved promoter
regions. For the experimentally-defined mouse promoter region,
analysis with classical vertebrate profiles of TF did not identify any
TFBS.

In order to find TFBS in mouse, universal protein binding
microarray data was used. UNIPROBE database (and its standard TF
binding algorithm) was used to find TFBS in experimentally-defined
mouse promoter sequence, using strict criterion of enrichment score
=0.49 [19].

Genomic DNA extraction

Genomic DNA was isolated from mammalian cell lines using
Wizard SV Genomic DNA Purification System (Promega, UK)
according to manufacturer’s instructions. Monkey DNA was extracted
from COS-7 cell line, dog from MDCK cell line and mouse from IMCD3
cell line. All cell lines were purchased from ADCC.

Fragment design and amplification

The genomic DNA sequence in the three test species was examined
and fragments homologous to the three human fragments identified.
In the mouse, where homology was limited, we initially looked for any
conserved TFBS, but there were none observed. Therefore, fragments
surrounding the Prpf31 TSS were designed arbitrarily, according to
possibility of PCR amplification in a difficult GC-rich region. In the dog,
where the homologous region was approximately 2000bp upstream of Prpf31 TSS, fragments were designed immediately adjacent to the TSS
also. The twelve regions of interest were amplified by PCR using KOD
polymerase (Novagen) and cloned into pGL3-basic vector (Promega,
UK) in both forward (indicated by +) and reverse (indicated by -)
strand orientation. Primers and PCR conditions can be seen in Table
6. In total, twelve regions were selected for assay by dual luciferase
reporter assay (Figure 2).

Table 6: Primer sequence and PCR conditions used for production of genomic fragments.

Dual luciferase reporter assays

The pGL3-reporter constructs were transfected into RPE-1 and
HeLa cell lines. Additionally, due to concerns about species-specific
transcription factor differences, mouse constructs were transfected
into IMCD3 cell lines, and dog constructs into MDCK cell lines. Dual
luciferase reporter assays were performed in quadruplicate, on three
separate occasions. A negative control (pGL3-basic) and positive
control (minimal thymidine kinase promoter, pTK) was assayed in
each experiment. The transfection protocol and dual-luciferase reporter
assay were performed as previously described [15]. Reporter assay data
was analysed by firstly standardizing for cell number, by calculating the
ratio of firefly luciferase (test) to renilla luciferase (control). This value
was then compared to the pTK values, as pTK is considered a goldstandard
basic promoter and, therefore, if a fragment has equivalent or
greater activity, it can be considered an active regulatory region.

Prpf31 expression studies

Whole eye and retina tissues were obtained from DBA/2, 129S2/
Sv and C57Bl/6J adult wild-type mice, ten animals from each strain
were used. The whole eye from the right side and the retina from the
contralateral side were collected from each animal. Total RNA was
extracted using TRIzol kit (Gibco BRL) according to the manufacturer’s
instructions. cDNA was prepared using the QuantiTect® Reverse
Transcription kit (Qiagen) and 1 μg of RNA as template for each reaction.
Real-time PCR was carried out with the GeneAmp 7500 System
(Applied Biosystem). The PCR reaction was performed using 1
μl cDNA, 12.5 ml SYBR Green Master Mix (Applied Biosystem) and
400 nM primer. Water was added to make a total reaction volume
of 20 μl. The PCR conditions were as follows: preheating, 50°C for 2
min and 95°C for 10 min; cycling, 40 cycles of 95°C for 15 s and 60°C for 1 min. Quantification results were expressed in terms of the cycle
threshold (Ct). The Ct values were averaged for each triplicate. Both
the Gapdh (F- GTATGACTCCACTCACGGCAAA; R- TTCCCATTCTCGGCCTTG)
and Hprt (F- GAAGAGCTACTGTAATGATCAG;
R- GCTGTACTGCTTAACCAGGG) were used as endogenous
controls (reference markers). Differences between the mean Ct values
of Prpf31 (F-TCGTGTGGACAGCTTCCATG; R- TTCTTCCGCTGCCCATCAAG)
and those of the reference genes were calculated as
ΔCt=CTPrpf31-CTHprt (or Gapdh). Relative fold changes in expression levels
were determined as 2-ΔΔCt, the Prpf31 expression data was normalised with the DBA/2 mouse strain.

Results

Bioinformatic analysis

The core promoter of the human PRPF31 gene had previously been
identified as Bi-P, the region at chr19:54618440-54619393 (hg19) and
the TFPT promoter was contained within this region (chr19:54618440-
54619133, hg19) [15]. Conservation of the defined regulatory region
was analyzed in several species from different lineages, showing a
remarkably low level of conservation (Figure 1). It was particularly
evident that chicken, Xenopus and zebrafish shared no homology
(defined as <25%) with the human region. In the mammalian lineage,
macaque and dog shared a high level of homology (defined as >50%)
with human, whereas mouse only had a low level homology over a very
short distance, the majority of the defined human promoter having no
homologous region in mouse. Interestingly, it was noted that although
the base sequence was conserved between dog and human, the gene
transcription start site (TSS) was different, meaning the homologous
sequence in dog was located some 2000bp from the canine Prpf31 TSS.

Figure 1: Evolutionary conservation of the Bi-P region, defined as the core promoter for PRPF31 in the human genome. The genome architecture of PRPF31 and TFPT
is illustrated, showing exon 1 and TSS of each gene. Degree of conservation is indicated by vertical height of peaks, with areas of significant evolutionary conservation
highlighted by a dotted black line. Yellow – non-coding exons, blue - coding exons, salmon pink - introns, green – transposable elements and repeats.

In light of these findings, three species were selected for study: C.
sabaeus, C. familiaris and M. musculus. It was thought that these species
would be interesting, as the green monkey had high homology to the
human promoter, the dog had homology in sequence but different
genome architecture and the mouse appeared to have different gene
regulation entirely.

Definition of core promoter in C. sabaeus

Three fragments from the C. sabaeus (green monkey) genome that
showed very high homology to the human active promoter elements
were tested by dual-luciferase reporter assay (Figures 2 and 3, Table 1).

Figure 2: Schematic representation of the genomic regions assayed by dual-luciferase reporter assay. The three fragments with defined reporter activity in human are illustrated, as well as the homologous regions in African green monkey, dog and mouse. The PRPF31 TSS is indicated with a solid arrow, the TFPT TSS with a dashed
arrow. Where appropriate, the percentage homology with the human fragment is indicated.

Figure 3: Results of dual-luciferase reporter assay using genomic sequence from C. sabaeus (green monkey) in HeLa cell line (A) and RPE- cell line (B).
The data is presented as the average ratio of pGL3-insert to pTK, together with
an error bar of ± one standard deviation, + refers to fragments tested in forward
strand (PRPF31) orientation, - to reverse strand (TFPT) orientation. The absolute
data values can be seen in Table 1.

The assay showed that P.31-Luc- had the strongest reporter activity
in the reverse strand (TFPT) orientation [2.29 ± 0.30 (HeLa); 1.85 ± 0.32 (RPE-1)], this confirmed that P.31-Luc was a core promoter with
moderate activity, controlling the expression of TFPT in monkey.
The TFPT promoter was, therefore, conserved between monkey and
human.

In the forward strand (PRPF31) orientation, both Bi-P+ and Δ2+
had strong promoter activity [Bi-P+: 3.91 ± 0.52 (HeLa); 5.12 ± 0.95
(RPE-1); Δ2+: 3.74 ± 0.33 (HeLa); 5.72 ± 0.86 (RPE-1)]. It was clear that
both fragments were capable of acting as strong promoter elements.
There was no significant difference between the two fragments and,
therefore, it was not possible to state unequivocally which was the
active core promoter element in vivo. It is likely, however, that Bi-P
is the promoter fragment, as Δ2 does not contain the gene TSS, and
would not, therefore, allow correct binding of RNA polymerase II. The
Bi-P+ and Δ2+ fragments both showed strong promoter activity in
human and, as such, the function of these two fragments was conserved
between human and green monkey.

Definition of core promoter in C. familiaris

Initially, two fragments immediately upstream to the dog PRPF31
TSS were designed and assayed, these fragments being homologous
to intron 1 of the human gene (termed S1 and S2). Luciferase assay
in HeLa, RPE-1 and MDCK cell lines showed that these fragments
possessed no luciferase activity (Figures 2 and 4A, Table 2).

Subsequently, the fragments that showed homology to the human
active promoter fragments – but located 2000bp from Prpf31 TSS –
were tested by dual luciferase reporter assay in RPE-1, HeLa and MDCK
cell lines (Figures 2 and 4B-D, Table 2). This showed that the P.31-Luc- fragment was also the core Tfpt promoter in dog, indeed acting as a
stronger promoter than that seen in human [6.16 ± 0.74 (HeLa); 2.40
± 0.41 (RPE-1); 4.98 ± 0.97 (MDCK)]. The fragment homologous to
the human PRPF31 promoter, Bi-P+, did not have strong promoter
activity in dog, with reporter activity less than, or very similar to, pTK
[0.48 ± 0.09 (HeLa); 0.91 ± 0.13 (RPE-1); 1.16 ± 0.14 (MDCK)]. This
suggests that the Bi-P+ fragment does not control the expression of Prpf31 in the dog.

Figure 4: Results of dual-luciferase reporter assay using genomic sequence from C. familiaris (domestic dog). The region immediately upstream to the gene transcription
start site were initially assayed (A), followed by regions located 2000bp upstream [(B)-Hela, (C) – RPE-1, (D) – MDCK]. The data is presented as the average ratio of
pGL3-insert to pTK, together with an error bar of ± one standard deviation, + refers to fragments tested in forward strand (PRPF31) orientation, - to reverse strand (TFPT)
orientation. The absolute data values can be seen in Table 2.

It was apparent, however, that the constituent elements of Bi-P+
(P.31-Luc + and Δ2+) had promoter activity, although this was variable
between the cell lines tested. In both HeLa and RPE-1 cell lines, Δ2+
had the highest promoter activity [4.71 ± 0.67 (HeLa); 2.58 ± 0.58
(RPE-1)], whereas P.31-Luc+ showed only slight activity [1.77 ± 0.42
(HeLa); 0.99 ± 0.30 (RPE-1)]. This situation was not observed in MDCK cell line, where P.31-Luc+ possessed strong promoter activity [5.01 ±
0.52 (MDCK)], although Δ2+ also displayed good reporter activity
[2.65±0.46 (MDCK)]. This suggested that the strong activation of
P.31-Luc+ requires the binding of a dog-species specific transcription
factor (TF), and that P.31-Luc is a true bi-directional promoter in C.
familiaris, controlling the expression of both Prpf31 and Tfpt.

Definition of core promoter in M. musculus

There was very little homology between the regions surrounding Prpf31 TSS in the mouse genome and the corresponding region in the
human genome. Of the three active human fragments (Bi-P, P.31-Luc
and Δ2), only Δ2 had a homologous region in the murine genome
(approximately 60% conservation). Therefore, the Δ2 fragment and
four additional fragments that shared no homology with human
regions (termed ψ1-4) were assayed by dual-luciferase reporter assay in
both forward- and reverse-strand orientations (Figures 2 and 5, Table
3).

Figure 5: Results of dual-luciferase reporter assay using genomic sequence from M. musculus (house mouse) in HeLa cells (A), RPE- cells (B) and IMCD3 cells (C).
The data is presented as the average ratio of pGL3-insert to pTK, together with an error bar of ± one standard deviation, + refers to fragments tested in forward strand
(PRPF31) orientation, - to reverse strand (TFPT) orientation. The absolute data values can be seen in Table 3.

In mouse, ψ1 acted as a true bidirectional promoter, controlling
the expression of Tfpt and Prpf31. The results were most clear in
IMCD3 (murine) cell line, where ψ1 had very strong reporter activity
in the forward strand orientation (6.61 ± 0.72) and the reverse strand
orientation (13.02 ± 0.75). The same effect was observed in HeLa cell
line (forward strand: 4.50 ± 0.60; reverse strand: 6.25 ± 0.62). In RPE-1
cell line, ψ1 had the strongest activity in the forward strand orientation
(2.85 ± 0.35); in the reverse strand orientation the situation was more complex, as three fragments had relatively strong reporter activity [Δ2:
(3.22 ± 0.26), ψ1: (3.76 ± 0.75), ψ2: (4.10 ± 0.22)]. This result might
be due to the different transcription factor profile in RPE-1 cells, and
the differences between human and murine transcription factors.
Given the clear result in IMCD3 cell line (the most realistic model of
the in vivo situation), it was concluded that ψ1 acted as a bidirectional
promoter controlling the expression of both Tfpt and Prpf31.

The ψ1 region in mouse shared no significant homology with any
human chromosomal region, with only very short regions (<54bp) of
imperfect alignment with human chromosomes 8, 14, 19, 20 and X
(Table 4).

Table 4: Regions in the human genome that are homologous to the experimentally defined mouse promoter, demonstrating minimal homology.

Prediction of transcription factor binding sites

A bioinformatic approach was taken to identify putative classical
TF binding sites (TFBS) within the experimentally-defined promoters
in monkey, dog and mouse. The transcription factor workspace of
PAZAR was used to look for TFBS in the characterized promoters
that showed conservation with human defined promoter. For mouse
prediction of TFBS by pairwise conservation between mouse and dog
was attempted.

As expected, there was a general overlap of TFBS between human
and monkey, for both PRPF31 and TFPT promoters (enriched in signal
transduction mechanism and transcription functions) (Figure 6A).
Moreover, it was observed that three TFBS (Myf, Gata1 and SP1) were
shared between human, monkey and dog (Figure 6A and 6B). It was not
possible, however, to identify any putative classical TFBS in the mouse
promoter using pairwise conservation between mouse and human or
mouse and dog (scanning for standard vertebrate transcription factor
profiles of Jaspar, Transfac & Oreganno). It was also observed that the
human promoter region was enriched with strong H3K4Me3 mark for
7 ENCODE cell lines assayed for this histone methylation (Figure 6).

Figure 6: Evolutionary conserved transcription factor binding sites (TFBS) in the experimentally-defined promoters of PRPF31 and TFPT in monkey (A) and dog
(B). TFBS conserved between all three species (human, monkey and dog) are underlined in red on (A). ORCA conserved regions highlight areas with high level of
evolutionary conservation between human and the test species. The ENCODE derived H3K4Me3 marks indicate areas that are often found in, or near, promoters.

As a conservation based approach could not be used in mouse,
due to lack of homology between mouse and human sequence, an
analysis was performed using universal protein binding microarray
(PBM) data, to identify putative TFBS in the experimentally-defined
murine bidirectional promoter (ψ1). A strict threshold of 0.49 TF
enrichment was used and all mouse PBM experiments in UNIPROBE
database were searched. A range of TF having strong binding affinity
to oligonucleotides of mouse promoter sequence were defined (Figure
7, Table 5). Amongst these, some TF classes were computationally
predicted to bind to characterized promoter in monkey and dog (e.g. TCFE2A, zinc finger family, NR2F). However, analysis of functional
enrichment showed that TFs binding to the mouse promoters were
enriched in purine metabolism and also Hox cluster genes (which
are important during development and homeostasis). These findings
support the divergent evolution of promoter sequences of human and
mouse, by gain of a new function in the mouse lineage.

In human populations, PRPF31 displays differentially expressed wild-type alleles, with highly expressed alleles providing protection
against the clinical manifestation of PRPF31-associated adRP. We
sought to analyze whether there was variable expression of Prpf31 in M. musculus. Real time qPCR experiments were performed, to
quantify Prpf31 expression levels in mouse eyes and retinas. In order
to avoid interference due to the genetic background or the age, animals
belonging to three different wildtype mouse strains (DBA/2, 129S2/Sv
and C57Bl/6J) and of the same age (8 weeks old) were analyzed. To
reach a statistical significant number of tested individuals, thirty mice
(ten mice for each strain) were analyzed. Prpf31 expression level was
tested in the eye and the contralateral retina of each mouse. Experiments
were performed using Hprt and Gapdh genes as endogenous control.
There was no statistically significant difference in Prpf31 expression
levels between the three mouse strains, either comparing eye or
retinal cDNAs (Figure 8). Overall, our data suggest that in the mouse
population there is no differential expression of Prpf31 alleles.

Discussion

The aim of this investigation was to identify and characterize the
core promoters controlling the expression of PRPF31 and TFPT in
three species, green monkey (C. sabaeus), domestic dog (C. familiaris)
and house mouse (M. musculus).

In green monkey, the core promoter of TFPT was defined as a
fragment (P.31-Luc) spanning -354 to +355 relative to the TFPT TSS,
with comparatively weak promoter activity. It was more difficult to
define the PRPF31 promoter in monkey, as both Bi-P+ and Δ2+ had
strong, and very similar, promoter activity. It was, however, considered
unlikely that Δ2 fragment is the true core promoter element of PRPF31, as it does not flank the TSS. It is apparent, however, that this fragment
is capable of acting as a promoter in vitro and is likely, therefore, to
harbour a RNA polymerase II binding site and other TFBS. As Bi-P spans the PRPF31 TSS (-406 to
+584), it is more likely that Bi-P is the in vivo promoter, as this would
allow correct binding of the RNA polymerase II. The two defined core
promoters, P.31-Luc and Bi-P, are homologous to the experimentallydefined
human promoters [15]. Furthermore, there were a large number
of evolutionarily conserved TFBS between human and monkey species.
This was to be expected, as the green monkey fragments share >90%
homology and there has, therefore, been conservation of the active
promoter elements.

In dog, P.31-Luc was defined as a true bidirectional promoter,
controlling the expression of both Prpf31 and Tfpt. P.31-Luc spanned
-510bp to +208bp relative to the Tfpt TSS in the dog genome, and had
strong promoter activity in this orientation in the three tested cell lines.
The fragment shared 73% homology with the human TFPT promoter,
so both the sequence and the function of this region were conserved
between the domestic dog and humans.

The region immediately upstream to canine Prpf31 TSS had no
reporter activity; but, rather, a long-range promoter was shown to
control the expression of Prpf31 in dog. P.31-Luc was defined as the
canine core promoter, and spanned -2580 to -1857 relative to the Prpf31 TSS (genomic co-ordinates chr1:103068699-103069421, canFam3).
Long-range promoter elements bind RNA polymerase II at the TSS (in
the same manner as canonical promoters), but distally bound TFs are
later brought into close apposition to the gene TSS by DNA looping, this allowing activation of the RNA polymerase II complex. It is unclear
why the sequence that is homologous to the primate exon 1 is not
transcribed in the canine lineage.

Interestingly, the Bi-P fragment, which shared 68% homology with
the human PRPF31 core promoter did not show reporter activity. The
difference between humans and dog indicates that the functional TFBS
in Bi-P have been lost in dog, or that new functional sites have evolved
since the divergence of the primate and canine lineages; the latter is
more likely, as functional domains tend to have a positive selection
pressure and are, therefore, rarely lost through evolution. Analysis
of TFBS identified three binding sites that were conserved between
human, monkey and dog, all of which are located within the human/
canine P.31-Luc portion, which correlates with the experimental
findings.

Furthermore, it should be noted that the human CNOT3 binding
site was not conserved in either monkey or dog, despite the higher level
of homology between the three species. It remains to be seen whether
this is reflected in less variable PRPF31 expression levels in wild
populations of these species, given that variable CNOT3 expression
was described as an important modulator of PRPF31 expression in the
human genome [19]. Another interesting finding of the TFBS prediction
was the finding of a conserved NR2E3 binding site between human
and monkey. NR2E3 is a transcription factor that plays an important
role in developmental differentiation of the photoreceptors and, after
development, is specifically expressed in post-mitotic photoreceptors
[20]. As such, regulation of PRPF31 by this factor might be part of the
explanation of the retina-specific phenotype of PRPF31 mutations.

A mouse fragment, ψ1 (chr7:3629316-3630581, mm10), was
defined as a bidirectional promoter controlling the expression of both
Tfpt and Prpf31. It was notable that the murine Prpf31 5' region shared
little homology with the human Prpf31 5' region; indeed, the region that
controlled expression of Prpf31 in mouse has no homologous region in
man. Bioinformatic analysis of conserved TFBS demonstrated that the
mouse promoter was not regulated by any shared putative TFs with
either human or dog. Instead, the mouse bidirectional promoter was
enriched with TFBS for Hox family TFs and purine metabolism TFs,
indicating significant divergent evolution in the mouse lineage.

It is necessary to speculate on the bidirectional gene architecture
of the PRPF31-TFPT gene pair, which is conserved throughout
the mammalian lineage, as well as in other vertebrate species, such
as chicken, Xenopus, anole lizard, Pelodiscus turtle and even the
coelacanth. The phenomenon of bidirectional gene pairs has been
observed across most genomes, including yeast, nematode, fish and
mammalian and it has been estimated that up to 10% of human
genes exist in this divergent arrangement [21]. Bidirectional genes
are controlled by a bidirectional promoter and this might allow cotranscription
of the two genes, in a way similar to the prokaryotic
operon. Bidirectional promoters are characterized by CpG islands, that
overlap the exon 1 of both genes and this strongly suggests that the
level of gene expression in bidirectional gene pairs is controlled by CpG
methylation [21,22]. It is unclear why the bidirectional TFPT-PRPF31 gene pair has arisen, given that the two genes share neither protein
function nor temporal expression. PRPF31 is a ubiquitously expressed
spliceosome component, whilst TFPT is involved in p53-independent
cellular apoptosis and is thus mainly active under conditions of cellular
stress. It is difficult to imagine a shared selection pressure that might
have influenced the gene architecture of these two very different genes.
It should be noted, however, that during cellular stress the splicing
machinery genes are down-regulated and, as such, the bidirectional
gene architecture might bear relevance to the complex changes in gene
expression that occur in situations of cellular stress [23].

In this work, it was also demonstrated that there is not variable
expression of Prpf31 in a population of mice from three different
strains. This suggests that the differential expression of Prpf31 has
arisen after the evolutionary divergence of rodent and primate lineages.
It could be inferred that the different 5’ architecture of the two genes
is responsible for the lack of differential expression in the mouse. It
has been demonstrated that one major factor that determines human PRPF31 expression level is repression of transcription by binding of
CNOT3 to the PRPF31 core promoter [9]. As the CNOT3 binding site
is not conserved between mouse and human, it follows that differential
gene expression is not observed in mouse populations. The different
gene regulation is a plausible explanation of why mouse models of
human Prpf31 mutations have failed to yield a disease phenotype
[11,12]. Furthermore, other cis-acting factors within the PRPF31 5’
region that are present in human, but not in mouse, might contribute
to the observed phenotypic differences.

It appears then, that in evolutionary terms, the high-expressing
allele is older and, since the divergence of rodents and primate lineages,
a lower-expressing allele has evolved. This raises complex evolutionary
questions that will need to be addressed through systematic
bioinformatic analysis of phylogenetic and genomic sequencing data.
These analyses might lead to a deeper understanding of this unusual
situation, whereby there appears to be rapidly-evolving control of an
evolutionarily-conserved gene, with different regulatory mechanisms
in relatively closely related species.

Acknowledgements

The authors are grateful for research support from the Rosetrees Trust,
Butterfield Trust (Bermuda), Foundation Fighting Blindness (USA), Fight for
Sight, RP Fighting Blindness and the Special Trustees of Moorfields Eye Hospital,
London, UK.